Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole

Abstract

Clotrimazole (CLT) is a widely used drug for the topical treatment of yeast infections of skin, vagina, and mouth. Common side effects of topical CLT application include irritation and burning pain of the skin and mucous membranes. Here, we provide evidence that transient receptor potential (TRP) channels in primary sensory neurons underlie these unwanted effects of CLT. We found that clinically relevant CLT concentrations activate heterologously expressed TRPV1 and TRPA1, two TRP channels that act as receptors of irritant chemical and/or thermal stimuli in nociceptive neurons. In line herewith, CLT stimulated a subset of capsaicin-sensitive and mustard oil-sensitive trigeminal neurons, and evoked nocifensive behavior and thermal hypersensitivity with intraplantar injection in mice. Notably, CLT-induced pain behavior was suppressed by the TRPV1-antagonist BCTC [(N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide)] and absent in TRPV1-deficient mice. In addition, CLT inhibited the cold and menthol receptor TRPM8, and blocked menthol-induced responses in capsaicin- and mustard oil-insensitive trigeminal neurons. The concentration for 50% inhibition (IC50) of inward TRPM8 current was approximately 200 nM, making CLT the most potent known TRPM8 antagonist and a useful tool to discriminate between TRPM8- and TRPA1-mediated responses. Together, our results identify TRP channels in sensory neurons as molecular targets of CLT, and offer means to develop novel CLT preparations with fewer unwanted sensory side effects.

Figure 1.

Effects of clotrimazole on calcium responses mediated by heterologously expressed TRPV1, TRPA1 and TRPM8. A, Average ratiometric [Ca²⁺]_i responses to 5 μm CLT and 100 nm capsaicin in TRPV1-transfected HEK cells loaded with Fura-2 AM (red trace; n = 8). In the same field, nontransfected cells did not respond to either stimuli (black trace; n = 10). B, Ratiometric [Ca²⁺]_i responses to 5 μm CLT and 20 μm MO in TRPA1-transfected HEK cells. Recordings from seven individual cells (red traces) are shown to illustrate the variability in the time course of the CLT responses. In the same field, nontransfected cells did not respond to either stimuli (black traces). C, Average ratiometric [Ca²⁺]_i responses to 100 μm menthol in the presence and absence of 10 μm CLT in TRPM8-transfected HEK cells (red trace; n = 10) and nontransfected cells in the same field (black trace; n = 5).

Figure 2.

Clotrimazole activates TRPV1-mediated whole-cell currents in HEK cells. A, Time course of the development of inward (at −50 mV) and outward (at +50 mV) TRPV1 current after application of 5 μm CLT. B, Current–voltage relations obtained during 200-ms voltage ramps from −50 to +50 mV applied at the time points indicated in (A). C, Comparison of the time course of TRPV1 current development after application of 1, 3, and 10 μm clotrimazole. D, Normalized dose–response curve for the CLT-induced TRPV1 current development at the indicated voltages. E, Currents elicited by 200 ms voltage steps ranging from −120 to +160 mV before (left) and during (right) addition of 3 μm CLT. F, Activation curves obtained from steady-state currents in the absence (filled circles) and presence of 3 μm CLT (open circles).

Figure 3.

Clotrimazole activates TRPA1-mediated whole-cell currents in CHO cells. A, Time course of the development of inward (at −75 mV) and outward (at +75 mV) TRPA1 currents after application of 5 μm CLT in control (Ca²⁺-free) extracellular solution. B, Current–voltage relations obtained during 500 ms voltage ramps from −150 to +150 mV at the time points indicated in A. C, Currents evoked in response to 400 ms voltage steps ranging from −150 to 150 mV followed by an invariant step to −150 mV before and during application of 7 μm CLT. D, Peak inward tail currents at −150 mV in the absence (filled circles) and presence of 7 μm CLT (open circles). The gray line represents the data obtained in the absence of CLT, shifted to the left by 110 mV. E, Time course of TRPA1 current development and desensitization after application of 5 μm clotrimazole in the presence of extracellular Ca²⁺ (5 mm; black line). A time course obtained in Ca²⁺-free solution (gray; same data as in Fig. 3A) is shown for comparison. F, Dose–response curve for the CLT-induced inward and outward TRPA1 current.

Figure 4.

Clotrimazole inhibits TRPM8-mediated whole-cell current in transfected HEK cells. A, Time course of inward (at −50 mV) and outward (at +50 mV) TRPM8 current activation by 100 μm menthol and its reversible inhibition by 10 μm CLT. B, Current–voltage relations obtained during 200 ms voltage ramps from −50 to + 50 mV at the time points indicated in A. C, Dose–response curve for the inhibition of menthol induced TRPM8 current by CLT at −50 mV (filled square) and +50 mV (open circles). D, Currents elicited in response to 100 ms voltage steps ranging from −120 to +160 mV in presence of 100 μm menthol, before (left) and during (right) addition of CLT. E, Activation curves obtained from steady-state currents for menthol-induced currents in the absence (filled circles) and presence (open circles) of 1 μm clotrimazole. F, Change in V_1/2 as a function of CLT concentration.

Figure 5.

Clotrimazole responses in mouse trigeminal neurons. A, Ratiometric [Ca²⁺]_i measurements from Fura-2 AM-loaded neurons from wild type mice showing different types of responses to 10 μm CLT, 1 μm capsaicin (Caps) and 60 mm K⁺. As depicted by circles (inset), CLT responses were only observed in capsaicin-sensitive neurons. B, Representative ratiometric [Ca²⁺]_i responses to 10 μm CLT, 100 μm capsaicin, 100 μm menthol (Men), 100 μm MO, and 60 mm K ⁺ in neurons (n = 83) from TRPV1^−/− mice. As depicted by circles (inset), CLT responses were only observed in mustard oil sensitive neurons. C, Percentage of wild-type and TRPV1^−/− neurons responding to capsaicin, CLT, and mustard oil. D, Cumulative probability plot of the responses to 1 μm capsaicin (red) and 10 μm CLT (green) in wild-type (solid lines) and TRPV1^−/− (dashed lines) neurons.

Figure 6.

Clotrimazole distinguishes between TRPM8- and TRPA1-mediated menthol-responses. A, Whole-cell currents in HEK cells transfected with TRPM8 illustrating the reversible inhibition of responses to menthol (100 μm) by CLT(10 μm). B, Ratiometric [Ca²⁺]_i responses to menthol (100 μm) in an MO-insensitive sensory neuron in the absence and presence of CLT (10 μm). C, Mean responses in menthol-sensitive, MO-insensitive sensory neurons (n = 15) to a stimulation protocol as in B, illustrating the strong inhibition (95 ± 2% block) of the menthol response by CLT. D, Same as in A, but for HEK cells transfected with TRPA1, illustrating the reversible potentiation of menthol responses by 10 CLT. E, Same as in B, but in MO-sensitive TRPV1^−/− sensory neurons. Two traces are shown, illustrating two distinct patterns of responses in these cells. A cell with a small response to a first menthol application (black trace; Δratio, 0.05) exhibited a strongly potentiated response to the combination menthol plus CLT. In contrast, CLT-induced potentiation of the menthol response was not observed in a cell with a robust response to a first menthol application (gray trace; Δratio, 0.65). F, Mean responses in MO-sensitive TRPV1^−/− sensory neurons (n = 15) to a stimulation protocol as in E.

Figure 7.

Intraplantar injection of Clotrimazole evokes pain and thermal hyperalgesia in mice. A, Duration of nocifensive behavior in the first 10 min after intraplantar injection of vehicle, CLT or CLT plus BCTC in wild-type and TRPV1^−/− mice. B, Change in response latency on a hot plate (55°C) 10–15 min after intraplantar injection of vehicle or CLT wild-type and TRPV1^−/− mice.